As the prevalence of Diabetes Mellitus increases, the need for recognizing the major cellular and molecular processes that underlie progression of the disease becomes more urgent. It is becoming increasingly clear that in patients with type 2 diabetes, P-cells are unable to fully compensate to maintain euglycemia overtime. 3-cell deficiency is preceded by a period in which p-cells are still viable but exhibit defective insulin storage/secretion. The endoplasmic reticulim (ER) becomes packed with proinsulin while production of normal insulin declines. This "golden period" before p-cell demise is of particular interest since it may highlight reversible processes that could be attacked both scientifically and therapeutically. A currently favored hypothesis is that proinsulin misfolding exacerbates 3-cell dysfunction, ultimately decreasing pancreatic insulin content and 3-cell mass. As in the Akita mouse, point mutations in proinsulin that alter its folding result in persistent congenital diabetes despite abundant nonmutant proinsulin available from wild-type alleles (autosomal dominant inheritance). In the K99 years, I have developed a novel approach to study insulin content and 3-cell mass in live animals, as well as to explore potential interactions between normal and misfolded proinsulin. Human proinsulin labeled with mid-region green fluorescent protein (GFP) can mimic the normal folding, trafficking, processing, and secretion of the native proinsulin in cell lines as well as in a transgenic mouse line called hProins-CpepGFP. In the ROO grant I am working on a new model in which the Akita mutation is introduced directly into the hProins-CpepGFP transgene (in cis) to study the fate of the misfolded proinsulin. I have also developed a new transgenic mouse line bearing GFP tagged Akita proinsulin that also bears an additional point mutation eliminating the partner (B7) free thiol group to be monitored for development of dominant negative diabetes. I plan to breed the hProinsC(A7)Y-CpepGFP line against Leptin receptor deficient db-/- mice in order to examine pathogenic synergies between proinsulin misfolding and increased insulin secretory demand. These studies will explore potentially reversible pathways leading to type 2 diabetes.